The steroid-binding properties of recombinant glucocorticoid receptor: a putative role for heat shock protein hsp90.
The steroid-binding domain of the human glucocorticoid receptor was expressed in Escherichia coli either as a fusion protein with protein A or under control of the T7 RNA polymerase promoter. The recombinant proteins were found to bind steroids with the normal specificity for a glucocorticoid receptor but with reduced affinity (Kd for triamcinolone acetonide approximately 70 nM). Glycerol gradient analysis of the E. coli lystate containing the recombinant protein indicated no interaction between the glucocorticoid receptor fragment and heat shock proteins. However, synthesis of the corresponding fragments of glucocorticoid receptor in vitro using rabbit reticulocyte lystate resulted in the formation of proteins that bound triamcinolone acetonide with high affinity (Kd 2nM). Glycerol gradient analysis of these proteins, with and without molybdate, indicated that the in vitro synthesised receptor fragments formed complexes with hsp90 as previously shown for the full-length rat glucocorticoid receptor. Radiosequence analysis of the recombinant steroid-binding domain expressed in E. coli and affinity labelled with dexamethasone mesylate identified binding of the steroid to Cys-638 predominantly. However, all cysteine residues within the steroid-binding domain were affinity labelled to a certain degree indicating that the recombinant protein has a structure similar to the native receptor but more open and accessible.
Ohara-Nemoto
Y
Y
Department of Medical Nutrition, Karolinska Institutet, Huddinge University Hospital, Sweden.
Strömstedt
P E
PE
Dahlman-Wright
K
K
Nemoto
T
T
Gustafsson
J A
JA
Carlstedt-Duke
J
J
D000345:Affinity Labels; D000595:Amino Acid Sequence; D002499:Centrifugation, Density Gradient; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D003907:Dexamethasone / Q000031:analogs & derivatives / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000235:genetics; D014222:Triamcinolone Acetonide / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D000345:Affinity Labels; D006360:Heat-Shock Proteins; D010446:Peptide Fragments; D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; D003907:Dexamethasone; D012321:DNA-Directed RNA Polymerases; D014357:Trypsin; D014222:Triamcinolone Acetonide; C031710:dexamethasone 21-methanesulfonate